GENOMICS
V&58-62
(1992)
Assignment of the Gene (RLBPI) for Cellular RetinaldehydeBinding Protein (CRALBP) to Human Chromosome 15q26 and Mouse Chromosome 7 ROBERT 5. SPARKES,* CAMILLA HEINZMANN, * STEVE COLDFLAM,t TRACY KOJIS,* JOHN C. SAARI,* T. MOHANDAS,~ IVANA KLISAK,* J. BRONWYN BATEMAN,” AND JOHN W. Cfwmt *Department of Medicine, UCLA School of Medicine and Center for the Health Sciences, Los Angeles, California 90024; t W. Alton Jones Cell Science Center, Lake Placid, New York 12946; *Department of Ophthalmology, University of Washington, Seattle, Washington 98196; §Division of Medical Genetics, Harbor/UCLA Medical Center, Torrance, California 90509; and “‘Jules Stein Eye Institute and Department of Ophthalmology, UCLA School of Medicine and Center for the Health Sciences, Los Angeles, California 90024 Received
March
25, 1991;revised
September
3, 1991
interaction with visual cycle enzymes in the retinol pigment epithelium. Because CRALBP is potentially important in maintaining normal physiological function in the retina, an absent or genetically altered protein may be associated with retinal disease. In a continuing effort to understand the role CRALBP may play in vision disorders, we report here the determination of the chromosomal location of the mouse and human genes encoding CRALBP.
Cellular retinaldehyde-binding protein (CRALBP) has properties that suggest that it is involved in the visual process and, therefore, potentially with retinal diseases. A human cDNA probe has been used to map this gene to human chromosome 15q26 (somatic cell hybrids and in situ hybridization) and to mouse chromosome 7 by somatic cell hybrids. 0 1992 Academic Press. Inc.
INTRODUCTION MATERIALS
Cellular retinaldehyde-binding protein (CRALBP) is a 36-kDa water-soluble protein found only in retina and pineal gland that carries 11-cis-retinaldehyde or 11-cis-retinol as physiological ligands (Saari et al, 1982). Several properties of CRALBP suggest that the protein is involved with the visual process. First, CRALBP has only been detected in tissues that respond to light. Second, the endogenous ligands carried by CRALBP are only known to function in vision. Third, CRALBP is able to select ll-cis-retinaldehyde from a mixture of vitamin A isomers and, relative to rhodopsin, protect it from photoisomerization, suggesting a protective role in the generation of 11-cis-retinoids (Saari and Bredberg, 1987). Finally, CRALBP interacts specifically with 11-cis-retinol dehydrogenase of retinol pigment epithelium (Saari and Bredberg, 1982) and influences the partitioning of llcis-retinol between enzymatic oxidation or esterification in retinol pigment epithelium (Saari and Bredberg, 1990). A functional role for CRALBP consistent with these properties is that of a substrate carrier protein that solubilizes the retinoid and modulates its O&38-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
METHODS
Probe A 1317-bp human cDNA for CRALBP was used for the mapping studies (Crabb et al., 1988). It was labeled by oligonucleotide priming with 32P-labeled deoxynucleotides as described previously (Feinberg and Vogelstein, 1983). The probe was shown to crosshybridize with human and mouse DNA and was used for the somatic cell hybrid and in situ hybridization mapping studies. Somatic Cell Hybrids For human mapping, a panel of 17 mouse/human somatic cell hybrids was derived from the fusion of thymidine kinase deficient mouse cells (B82, GM 0347A) and normal male fibroblasts (IMR 91) as described previously (Mohandas et al., 1986). Cytogenetic analysis was carried out on a minimum of 30 Q-banded metaphases per hybrid clone. For mouse mapping, somatic cell hybrids were derived from the fusion of an established thymidine ki58
Inc. reserved.
AND
MAPPING
OF RLBPl
IN HUMANS
59
AND MICE
-8.3 21 1 21 2 21 3 22 22
1 2
22
3
m
23 21
-2.2
25
FIG. 1. Southern blot pattern with the RLBPl probe. The hybrid clear human bands are seen in the nel, and both were used for scoring
using the human/mouse panel DNA was cut with EcoRI. Two IMR91 (human parent) chanthe human gene in the hybrids.
nase-deficient Chinese hamster cell line, RJK, and spleen cells from BALB/c (series 112) or AKR (series 118) mice. A mixture of the parental cells in suspen-
26
1
26 26
23 mi=
15 FIG. 3. Detail of human tion of the autoradiographic
chromosome grains.
15 showing
the distribu-
sion was treated with polyethylene glycol to induce cell fusion (Davidson and Gerald, 1976; Kennett, 1979). Independent hybrid clones were isolated in HAT medium. High-molecular-weight DNA was ex-
15 1 10 5 1
3 2 3
P
1
I
4
P’q
6
z
P 7
9
P
II
I
8
q
P’q
9
P 10
II q
ii
PI.2 1.2
15.5 -
6.69 13
14
15
16
17
1s
19
20
21
22
x
Y
CHROMOSOMES
FIG. 2. Full histogram for the in situ labeling of human metaphase chromosomes. These results are from 108 metaphases, 40 of which had a total of 67 grains; 18 are on chromosome 15 with the strongest concentration at band q26.
FIG. 4. Southern blot pattern using the hamster/mouse panel with the RLBPl probe. The hybrid DNA was cut with HindIII. The mouse 15.5kb band was used for scoring the mouse gene in the hybrids.
60
SPARKES
ET
TABLE Mapping
of Human
RLBPl
AL.
1 in Somatic Cell Hybrids
Human Hybrid clone 84-2 84-3 84-4 84-5 84-13 84-20 84-26 84-27 84-35 84-38 84-39 84-7 84-21 84-25 84-30 84-34 84-37 No. of discordant hybrids
RLPBl” + + + + + + + + + + +
-
1
2
3
4
5
6
-
+-
(+) : + + + + (+) + -+--++-+---++--+-++-++----++-+++---+-+++++ +-++-+++-++++++-++ +---+++------+++
+
++-+++++--++-+++++++-+-+t++-+++-+--+++-++++---++++-+++-+--+++-++++----++-++-++--
-
; -
++-+++--++-+-++--++--+ + + + + (+) + + +
6549
LI t, Indicates presence of the probe sequence. b f, Indicates presence in lo-30% of metaphases
7
+ +
8
9
10
11
-
-
+
-
+ +
-
-
(+) -
sequences
12
13
-
(+) + +-+-++-++--+ + + +
(1, +
14
15
16
17
18
19
20
21
22
x
Y
+
-
+
-
+
-
+
+
+
-
+
-
+ +
+ +
+
(l,
+ +
+ +
(+) ~
~ (+)
---+
(+)
-
+ ---
--
-
----+--f-+-+--+--f++ + + + + + + + (+)
8775 of the probe
chromosomesb
in the hybrid
-
+ + + -
+ + +
(+) (+) +
+ -
~ + + +
-
-
+ + + +
+ -
(+) -
(+) + +
+ + +
+ + +
-
(+) -
11
11
12
9
5
8
1
11
6
3
6
8
10
9
9
11
clone
determined
by the presence
of the human chromosome in greater than 30% of metaphases analyzed; -, indicates absence of the chromosome.
tracted from the hybrid clones as described in Sparkes et al. (1986), at which time they were also analyzed cytogenetically using Q banding and fluorescent staining with Hoechst 33258 sequentially (Kozak et al., 1977).
In Situ Hybridization The CRALBP DNA probe was labeled by oligonucleotide priming with 3H-labeled deoxynucleotides to a specific activity of about 3 X lo8 CPM/pg. In situ hybridization to normal human chromosomes followed the method of Harper and Saunders (1981) as modified by Cannizzaro and Emanuel (1984). Slides were exposed for 1 week and all silver grains on or touching chromosomes were scored.
analyzed;
of the human (+),
indicates
band.
-, Indicates
presence
the absence
of the chromosome
the only chromosome with one discordancy while all other chromosomes had at least three discordancies. The reason for the one false negative result with hybrid 84-20 is probably the fact that this hybrid does contain 8% of the cells with chromosome 15. The results of the in situ hybridization studies are seen in Figs. 2 and 3. The full histogram confirms the assignment of the gene to chromosome 15. The peak of the label is at 15q26. The results of the mouse mapping, seen in Fig. 4 and Table 2, indicate assignment of the gene to mouse chromosome 7 based on no discordancies observed for this chromosome while all other chromosomes showed at least one discordancy.
DISCUSSION RESULTS The chromosomal location of the RLBPl gene was determined by comparing the human chromosome content of the hybrid lines with human-specific bands on the Southern blot probed with RLBPl cDNA. The results of these studies are seen in Fig. 1 and Table 1. The gene is assigned to chromosome 15 since this was
The somatic cell hybrid studies presented here indicate that the human gene for RLBPl is on human chromosome 15. This is confirmed by the in situ hybridization analysis, which further maps the gene to 15q26. Using somatic cell hybridization analysis, the gene is mapped to mouse chromosome 7. No known retinal disease maps to these chromosomes. The ho-
MAPPING
OF
RLBPl
IN
HUMANS
TABLE Mapping
of Mouse RLBPl
RLBPl”
118-l 118-3 118-11 118-12 118-14 118-15 112-13 118-7-A 112-8 118-4-Q 130-17
+ + +
130-27B No. of discordant hybrids
+ + + + + -
1
2
3
61
MICE
2 in Somatic
Mouse Hybrid clone
AND
Cell Hybrids
chromosomes*
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
x
Y
+ + + + ++-+-+++++++++++ -+----+ ++++-++--+-++-+---+-+ +++--+++-+++-++++ ++ -++ ++++--++-+++++++++++------++------
(+)
+
+
+
+
+
+
+
+
+
+
+
+
(+)
+
+
+
+
+
+
+
+ (+) -
+ -
+ + +
-
+ -
(+I
+
(+I
+
-
(+)
+
-
+
(+I +
+ +
-
+ -
tt-
_
+ + -
(+) -
-
+ -
-
+ -
_-
+
+ -
+ +
+ + -
+ + -
-
+ -
+ + + +
(+) + + -
+ -
+ +
-
+ +
+ + -
-
-
+
(+)
-
-
(+)
-
+
-
+
-
-
-
+
+
-
-
+
-
-
+
D +, Indicates presence of the gene. * +, Indicates presence in lo-30% of metaphases
435465045443344447196 of the CRALBP
sequences
in the hybrid
clone
determined
of the human chromosome in greater than 30% of metaphases analyzed, -, indicates absence of the human chromosome.
mologies between human chromosome region 15q25 and 15q26 with mouse chromosome 7 are confirmed by these studies. No other known retinal specific genes map in these regions. An analysis of patients with retinitis pigmentosa and Usher syndrome type I using this probe failed to demonstrate any gross alteration of the CRALBP gene in affected patients or demonstration of cosegregation in a limited number of families with these hereditary eye diseases (Cotran et al., 1990). No other linkage studies with genes in this region are known to the authors. Nevertheless, because of the probable heterogeneity of patients with retinitis pigmentosa, further studies need to be carried out with the autosoma1 forms of retinitis pigmentosa to further evaluate alterations in this gene in this group of diseases. The structure of the human gene encoding RLBPl has recently been established (Crabb et aZ., 1991a). The human RLBPl gene is composed of eight exons ranging in size from 79 to 201 nucleotides and of seven introns within about 13 kilobases of DNA. As a preliminary approach to identifying functional domains and protein recognition sites in CRALBP, a low-resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis (Crabb et al., 1991b). These and other ongoing studies, including continued disease linkage efforts, should provide a more definitive understanding of the normal and pathophysiological role of this protein.
by the presence analyzed,
of the mouse (+),
indicates
band, presence
-, indicates
absence
of the chromosome
ACKNOWLEDGMENTS This work was supported in part by United States Public Health Service Grant EY06603 (J.W.C.) andGordon Gund/National Retinitis Pigmentosa Foundation Grant (R.S.S., C.H., T.K., and J.B.B.).
REFERENCES 1.
CANNIZZARO, L. A., AND EMANUEL, B. S. (1984). An improved method for G-banding chromosomes after in situ hybridization. Cytogenet. Cell Genet. 38: 308309.
2.
COTRAN, P. R., RINGENS, P. J., CRABB, J. W., BERSON, E. L., AND DRYJA, T. P. (1990). Analysis of the DNA of patients with retinitis pigmentosa with a cellular retinaldehyde binding protein cDNA. Exp. Eye Res. 51: 15-19. CRABB, J. W., GOLDFLAM, S., HARRIS, S. E., AND SAARI, J. C. (1988). Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures. J. Btil. Chem. 263: 18,688 18,692.
3.
4.
5.
6.
7.
CRABB, J. W., INTRES, R., GOLDFLAM, S., AND COOK, J. R. (1991a). The structure of the human gene encoding cellular retinaldehyde-binding protein. FASEB J. 5: A1173. CRABB, J. W., GAUR, V. P., GARWIN, G. G., MARX, S. V., CHAPLINE, C., JOHNSON, C. M., AND SAARI, J. C. (1991b). Topological and epitope mapping of the cellular retinaldehyde-binding protein. J. Biol. Chem. 266: 16,674-16,683. DAVIDSON, R. L., AND GERALD, P. S. (1976). Improved techniques for the induction of mammalian cell hybridization by polyethylene glycoi. Somat. Cell Genet. 2: 165-176. FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
62
SPARKES
8. HARPER, M. E., AND SAUNDERS, G. F. (1981). Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83: 431-439. 9. KENNETT, R. H. (1979). Cell fusion. In. “Methods in Enzymology” (W. B. Jakoby and I. H. Pastan, Eds.), Vol. 58, pp. 345358, Academic Press, New York. 10. KOZAK, C. A., LAWRENCE, J. B., AND RUDDLE, F. H. (1977). A sequential staining technique for the chromosomal analysis of interspecific mouse/hamster and mouse/human somatic cell hybrids. Exp. Cell Res. 105: 109-117. 11. MOHANDAS, T., HEINZMANN, C., SPARKES, R. S., WASMUTH, J., EDWARDS, P., AND Lusrs, A. J. (1986). Assignment of human 3-hydroxy3methylglutaryl coenzyme A reductase gene to q13-q23 region of chromosome 5. Somat. Cell Mol. Genet. 12: 89-94.
12. SAARI, J. C., BREDBERG, D. L., AND GARWIN, G. G. (1982). Identification of the endogenous retinoids associated with
ET
13. 14. 15. 16.
AL.
three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium. J. Bill. Chem. 267: 13,32913,333. SAARI, J. C., AND BREDBERG, D. L. (1982). Enzymatic reduction of ll-&-retinal bound to cellular retinal-binding protein. Biochim. Biophys. Acta. 716: 266-272. SAARI, J. C., AND BREDBERG, D. L. (1987). Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina. J. Biol. Chem. 262: 7618-7622. SAARI, J. C., AND BREDBERG, D. L. (1990). Modulation of visual cycle enzyme reaction parameters by retinoid-binding proteins. Inuest. Ophthulmol. Vis. Sci. (Suppl.) 31: 111. SPARKES, R. S., MOHANDAS, T., NEWMAN, S. L., HEINZMANN, C., KAUFMAN, D., ZOLLMAN, S., LEVEILLE, P. J., ToBIN, A. J., AND MCGINNIS, J. F. (1986). Assignment of the rhodopsin gene to human chromosome 3. Inuest. Ophthulmd. Vi+-. Sci. 127:
1170-1172.
V&58-62
(1992)
Assignment of the Gene (RLBPI) for Cellular RetinaldehydeBinding Protein (CRALBP) to Human Chromosome 15q26 and Mouse Chromosome 7 ROBERT 5. SPARKES,* CAMILLA HEINZMANN, * STEVE COLDFLAM,t TRACY KOJIS,* JOHN C. SAARI,* T. MOHANDAS,~ IVANA KLISAK,* J. BRONWYN BATEMAN,” AND JOHN W. Cfwmt *Department of Medicine, UCLA School of Medicine and Center for the Health Sciences, Los Angeles, California 90024; t W. Alton Jones Cell Science Center, Lake Placid, New York 12946; *Department of Ophthalmology, University of Washington, Seattle, Washington 98196; §Division of Medical Genetics, Harbor/UCLA Medical Center, Torrance, California 90509; and “‘Jules Stein Eye Institute and Department of Ophthalmology, UCLA School of Medicine and Center for the Health Sciences, Los Angeles, California 90024 Received
March
25, 1991;revised
September
3, 1991
interaction with visual cycle enzymes in the retinol pigment epithelium. Because CRALBP is potentially important in maintaining normal physiological function in the retina, an absent or genetically altered protein may be associated with retinal disease. In a continuing effort to understand the role CRALBP may play in vision disorders, we report here the determination of the chromosomal location of the mouse and human genes encoding CRALBP.
Cellular retinaldehyde-binding protein (CRALBP) has properties that suggest that it is involved in the visual process and, therefore, potentially with retinal diseases. A human cDNA probe has been used to map this gene to human chromosome 15q26 (somatic cell hybrids and in situ hybridization) and to mouse chromosome 7 by somatic cell hybrids. 0 1992 Academic Press. Inc.
INTRODUCTION MATERIALS
Cellular retinaldehyde-binding protein (CRALBP) is a 36-kDa water-soluble protein found only in retina and pineal gland that carries 11-cis-retinaldehyde or 11-cis-retinol as physiological ligands (Saari et al, 1982). Several properties of CRALBP suggest that the protein is involved with the visual process. First, CRALBP has only been detected in tissues that respond to light. Second, the endogenous ligands carried by CRALBP are only known to function in vision. Third, CRALBP is able to select ll-cis-retinaldehyde from a mixture of vitamin A isomers and, relative to rhodopsin, protect it from photoisomerization, suggesting a protective role in the generation of 11-cis-retinoids (Saari and Bredberg, 1987). Finally, CRALBP interacts specifically with 11-cis-retinol dehydrogenase of retinol pigment epithelium (Saari and Bredberg, 1982) and influences the partitioning of llcis-retinol between enzymatic oxidation or esterification in retinol pigment epithelium (Saari and Bredberg, 1990). A functional role for CRALBP consistent with these properties is that of a substrate carrier protein that solubilizes the retinoid and modulates its O&38-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
METHODS
Probe A 1317-bp human cDNA for CRALBP was used for the mapping studies (Crabb et al., 1988). It was labeled by oligonucleotide priming with 32P-labeled deoxynucleotides as described previously (Feinberg and Vogelstein, 1983). The probe was shown to crosshybridize with human and mouse DNA and was used for the somatic cell hybrid and in situ hybridization mapping studies. Somatic Cell Hybrids For human mapping, a panel of 17 mouse/human somatic cell hybrids was derived from the fusion of thymidine kinase deficient mouse cells (B82, GM 0347A) and normal male fibroblasts (IMR 91) as described previously (Mohandas et al., 1986). Cytogenetic analysis was carried out on a minimum of 30 Q-banded metaphases per hybrid clone. For mouse mapping, somatic cell hybrids were derived from the fusion of an established thymidine ki58
Inc. reserved.
AND
MAPPING
OF RLBPl
IN HUMANS
59
AND MICE
-8.3 21 1 21 2 21 3 22 22
1 2
22
3
m
23 21
-2.2
25
FIG. 1. Southern blot pattern with the RLBPl probe. The hybrid clear human bands are seen in the nel, and both were used for scoring
using the human/mouse panel DNA was cut with EcoRI. Two IMR91 (human parent) chanthe human gene in the hybrids.
nase-deficient Chinese hamster cell line, RJK, and spleen cells from BALB/c (series 112) or AKR (series 118) mice. A mixture of the parental cells in suspen-
26
1
26 26
23 mi=
15 FIG. 3. Detail of human tion of the autoradiographic
chromosome grains.
15 showing
the distribu-
sion was treated with polyethylene glycol to induce cell fusion (Davidson and Gerald, 1976; Kennett, 1979). Independent hybrid clones were isolated in HAT medium. High-molecular-weight DNA was ex-
15 1 10 5 1
3 2 3
P
1
I
4
P’q
6
z
P 7
9
P
II
I
8
q
P’q
9
P 10
II q
ii
PI.2 1.2
15.5 -
6.69 13
14
15
16
17
1s
19
20
21
22
x
Y
CHROMOSOMES
FIG. 2. Full histogram for the in situ labeling of human metaphase chromosomes. These results are from 108 metaphases, 40 of which had a total of 67 grains; 18 are on chromosome 15 with the strongest concentration at band q26.
FIG. 4. Southern blot pattern using the hamster/mouse panel with the RLBPl probe. The hybrid DNA was cut with HindIII. The mouse 15.5kb band was used for scoring the mouse gene in the hybrids.
60
SPARKES
ET
TABLE Mapping
of Human
RLBPl
AL.
1 in Somatic Cell Hybrids
Human Hybrid clone 84-2 84-3 84-4 84-5 84-13 84-20 84-26 84-27 84-35 84-38 84-39 84-7 84-21 84-25 84-30 84-34 84-37 No. of discordant hybrids
RLPBl” + + + + + + + + + + +
-
1
2
3
4
5
6
-
+-
(+) : + + + + (+) + -+--++-+---++--+-++-++----++-+++---+-+++++ +-++-+++-++++++-++ +---+++------+++
+
++-+++++--++-+++++++-+-+t++-+++-+--+++-++++---++++-+++-+--+++-++++----++-++-++--
-
; -
++-+++--++-+-++--++--+ + + + + (+) + + +
6549
LI t, Indicates presence of the probe sequence. b f, Indicates presence in lo-30% of metaphases
7
+ +
8
9
10
11
-
-
+
-
+ +
-
-
(+) -
sequences
12
13
-
(+) + +-+-++-++--+ + + +
(1, +
14
15
16
17
18
19
20
21
22
x
Y
+
-
+
-
+
-
+
+
+
-
+
-
+ +
+ +
+
(l,
+ +
+ +
(+) ~
~ (+)
---+
(+)
-
+ ---
--
-
----+--f-+-+--+--f++ + + + + + + + (+)
8775 of the probe
chromosomesb
in the hybrid
-
+ + + -
+ + +
(+) (+) +
+ -
~ + + +
-
-
+ + + +
+ -
(+) -
(+) + +
+ + +
+ + +
-
(+) -
11
11
12
9
5
8
1
11
6
3
6
8
10
9
9
11
clone
determined
by the presence
of the human chromosome in greater than 30% of metaphases analyzed; -, indicates absence of the chromosome.
tracted from the hybrid clones as described in Sparkes et al. (1986), at which time they were also analyzed cytogenetically using Q banding and fluorescent staining with Hoechst 33258 sequentially (Kozak et al., 1977).
In Situ Hybridization The CRALBP DNA probe was labeled by oligonucleotide priming with 3H-labeled deoxynucleotides to a specific activity of about 3 X lo8 CPM/pg. In situ hybridization to normal human chromosomes followed the method of Harper and Saunders (1981) as modified by Cannizzaro and Emanuel (1984). Slides were exposed for 1 week and all silver grains on or touching chromosomes were scored.
analyzed;
of the human (+),
indicates
band.
-, Indicates
presence
the absence
of the chromosome
the only chromosome with one discordancy while all other chromosomes had at least three discordancies. The reason for the one false negative result with hybrid 84-20 is probably the fact that this hybrid does contain 8% of the cells with chromosome 15. The results of the in situ hybridization studies are seen in Figs. 2 and 3. The full histogram confirms the assignment of the gene to chromosome 15. The peak of the label is at 15q26. The results of the mouse mapping, seen in Fig. 4 and Table 2, indicate assignment of the gene to mouse chromosome 7 based on no discordancies observed for this chromosome while all other chromosomes showed at least one discordancy.
DISCUSSION RESULTS The chromosomal location of the RLBPl gene was determined by comparing the human chromosome content of the hybrid lines with human-specific bands on the Southern blot probed with RLBPl cDNA. The results of these studies are seen in Fig. 1 and Table 1. The gene is assigned to chromosome 15 since this was
The somatic cell hybrid studies presented here indicate that the human gene for RLBPl is on human chromosome 15. This is confirmed by the in situ hybridization analysis, which further maps the gene to 15q26. Using somatic cell hybridization analysis, the gene is mapped to mouse chromosome 7. No known retinal disease maps to these chromosomes. The ho-
MAPPING
OF
RLBPl
IN
HUMANS
TABLE Mapping
of Mouse RLBPl
RLBPl”
118-l 118-3 118-11 118-12 118-14 118-15 112-13 118-7-A 112-8 118-4-Q 130-17
+ + +
130-27B No. of discordant hybrids
+ + + + + -
1
2
3
61
MICE
2 in Somatic
Mouse Hybrid clone
AND
Cell Hybrids
chromosomes*
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
x
Y
+ + + + ++-+-+++++++++++ -+----+ ++++-++--+-++-+---+-+ +++--+++-+++-++++ ++ -++ ++++--++-+++++++++++------++------
(+)
+
+
+
+
+
+
+
+
+
+
+
+
(+)
+
+
+
+
+
+
+
+ (+) -
+ -
+ + +
-
+ -
(+I
+
(+I
+
-
(+)
+
-
+
(+I +
+ +
-
+ -
tt-
_
+ + -
(+) -
-
+ -
-
+ -
_-
+
+ -
+ +
+ + -
+ + -
-
+ -
+ + + +
(+) + + -
+ -
+ +
-
+ +
+ + -
-
-
+
(+)
-
-
(+)
-
+
-
+
-
-
-
+
+
-
-
+
-
-
+
D +, Indicates presence of the gene. * +, Indicates presence in lo-30% of metaphases
435465045443344447196 of the CRALBP
sequences
in the hybrid
clone
determined
of the human chromosome in greater than 30% of metaphases analyzed, -, indicates absence of the human chromosome.
mologies between human chromosome region 15q25 and 15q26 with mouse chromosome 7 are confirmed by these studies. No other known retinal specific genes map in these regions. An analysis of patients with retinitis pigmentosa and Usher syndrome type I using this probe failed to demonstrate any gross alteration of the CRALBP gene in affected patients or demonstration of cosegregation in a limited number of families with these hereditary eye diseases (Cotran et al., 1990). No other linkage studies with genes in this region are known to the authors. Nevertheless, because of the probable heterogeneity of patients with retinitis pigmentosa, further studies need to be carried out with the autosoma1 forms of retinitis pigmentosa to further evaluate alterations in this gene in this group of diseases. The structure of the human gene encoding RLBPl has recently been established (Crabb et aZ., 1991a). The human RLBPl gene is composed of eight exons ranging in size from 79 to 201 nucleotides and of seven introns within about 13 kilobases of DNA. As a preliminary approach to identifying functional domains and protein recognition sites in CRALBP, a low-resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis (Crabb et al., 1991b). These and other ongoing studies, including continued disease linkage efforts, should provide a more definitive understanding of the normal and pathophysiological role of this protein.
by the presence analyzed,
of the mouse (+),
indicates
band, presence
-, indicates
absence
of the chromosome
ACKNOWLEDGMENTS This work was supported in part by United States Public Health Service Grant EY06603 (J.W.C.) andGordon Gund/National Retinitis Pigmentosa Foundation Grant (R.S.S., C.H., T.K., and J.B.B.).
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